Combinatorial Therapeutic Potential of Stem Cells and Benzimidazol Derivatives for the Reduction of Liver Fibrosis

(1) Background: Liver fibrosis is currently one of the top ten causes of death worldwide. Stem cells transplantation using mesenchymal stem cells (MSCs) is an alternative therapy which is used in the place of organ transplant, due to the incapacity of stem cells to endure oxidative stress in the damage site, thus affecting the healing process. The present study aimed to enhance the therapeutic potential of MSCs using combined therapy, along with the novel synthetic compounds of benzimidazol derivatives. (2) Methods: Eighteen compound series (benzimidazol derivatives) were screened against liver fibrosis using an in vitro CCl4-induced injury model on cultured hepatocytes. IC50 values were calculated on the bases of LDH assay and cell viability assay. (3) Results: Among the eighteen compounds, compounds (10), (14) and (18) were selected on the basis of IC50 value, and compound (10) was the most potent and had the lowest IC50 value in the LDH assay (8.399 ± 0.23 uM) and cell viability assay (4.73 ± 0.37 uM). Next, these compounds were combined with MSCs using an in vitro hepatocytes injury culture and in vivo rat fibrotic model. The effect of the MSCs + compounds treatment on injured hepatocytes was evaluated using LDH assay, cell viability assay, GSH assay and real-time PCR analysis and immuno-staining for caspase-3. Significant reductions in LDH level, caspase-3 and apoptotic marker genes were noted in MSCs + compounds-treated injured hepatocytes. In vivo data also showed the increased homing of the MSCs, along with compounds after transplantation. Real-time PCR analysis and TUNEL assay results also support our study. (4) Conclusions: It was concluded that compounds (10), (14) and (18) can be used in combination with MSCs to reduce liver fibrosis.


Introduction
The liver serves as the digestive organ of all vertebrates; it detoxifies metabolites, synthesizes proteins and produces the biochemicals needed for both digestion and development [1]. Almost all medicines are recognized by the human body as foreign substances (xenobiotics), which then undergo a number of chemical reactions (metabolism) to make them suitable for elimination. Portal veins, which carry drugs and xenobiotics in a nearly undiluted form, supply the liver with around 75% of its blood directly from the digestive treatment, MSC transplantation can restore wounded liver tissue in experimentally infected rats without completely removing the hydatid cyst [25]. In light of the abovementioned literature, the study in this article was designed with the aim to evaluate-for the first time-the therapeutic potentials of novel benzimidazol derivatives against liver fibrosis and their combined therapeutic potential-along with MSCs-on damaged hepatocytes induced by CCl 4 in in vitro hepatocytes and also in an experimental rat model.

Culturing and Characterization of Rat Hepatocytes
Hepatocytes from male Wister rat liver were isolated using the Ferrigno (2010) twostep perfusion method [26], and hepatocytes viability was checked by trypan blue assay, which showed that 85-95% of hepatocytes were viable. The Ferrigno (2010) method was then used to isolate hepatocytes from male Wister rat liver and trypan blue assay were conducted to check the viability of hepatocytes, which show that 85-95% of the hepatocytes were viable. The morphology of the cells was noted under a phase-contrast microscope after 2 h and 24 h of culturing cells in an RPMI medium. After 2 h, the cells were binucleated in a spherical shape, similar to the morphology of normal hepatocytes. The cells were flattened and polygonal in shape after 24 h of culturing, as shown in Figure  S1. After culturing, hepatocytes were characterized at both the RNA and protein levels, using real-time PCR and immunochemistry for the hepatocytes-specific marker. Cultured cells exhibited hepatocyte-specific markers, i.e., albumin, CK-8 and CK-18 according to real-time PCR analysis. As an endogenous control, β actin was employed ( Figure S2A). Hepatocytes' ability to store glycogen is also crucial; hence, PAS-staining was used to assess the functional status of the cultured hepatocytes. After PAS-staining, the cells became purple, indicating that the cultured hepatocytes were also functionally active ( Figure S2B). To examine the protein expression of certain hepatic cell markers in the cultured cells, immunostaining was performed. DAPI was used to label the nuclei of the cells. Albumin, CK-8, CK-18 and AFP-positive cells were found after immunostaining ( Figure S2Ca-d).

Hepatocytes Injury Analysis
For the preparation of the CCl 4 -treated hepatocytes injury culture, rat hepatocytes cells were treated with 5mM of CCl 4 concentrations for 6 h [27]. LDH assay ( Figure S3A), trypan blue assay ( Figure S3B), GSH assay ( Figure S3C) and real-time PCR analysis ( Figure S3D) were used to examine the hepatic marker expression (CK-8 and albumin), anti-apoptotic (Bcl-xL) markers and apoptotic (BAX and caspase-3) markers showed the maximum level of CCl 4 -induced hepatocytes injury.

In Vitro Screening of Compounds
The novel compounds of benzimidazol derivatives 1-18 were screened against liver fibrosis [28]. In vitro CCl 4 -induced injury culture was used for screening. LDH assay and trypan blue assay were performed to find the viability of the cells ( Figure S4A,B), from which the IC50 values of the compounds were calculated. GSH/GSSH assay was also performed to calculate the total glutathione level ( Figure S4C). Among all of the compounds, compound (10) was found to be the most potent compound because it has the lowest IC50 value and the highest GSH/GSSG value. In addition to compound (10), other compounds found to be potent were (14) and (18). Therefore, these three compounds were selected for in vitro and in vivo study in combination with MSCs. Compound structures and names, along with the IC50 values, are shown in Table 1. compounds were selected for in vitro and in vivo study in combination with MSCs. Compound structures and names, along with the IC50 values, are shown in Table 1.  First, the hepatocytes were exposed to a CCl4 dose (5 mM for 6 h) followed by treatment with compounds (10), (14) and (18) for 24 h. After the compounds treatment, MSCs were co-cultured with hepatocytes in a Transwell system with and without compounds. The co-cultures of hepatocytes were divided into nine groups, i.e., normal, CCl4-treated, three groups of only compound-treated, MSCs-treated and three groups of MSCs + compounds-treated. A significant decrease in the level of LDH was seen in compounds-treated and MSCs + compounds-treated injured hepatocytes ( Figure 1A). However, treatment with MSCs + compounds, predominantly compound (10), showed a significantly higher decrease in cell injury than any other treatment group. Cell viability ( Figure 1B) and GSH level ( Figure 1C) were highly increased in MSCs + compounds (10)-treated hepatocytes. 16 First, the hepatocytes were exposed to a CCl4 dose (5 mM for 6 h) followed by treatment with compounds (10), (14) and (18) for 24 h. After the compounds treatment, MSCs were co-cultured with hepatocytes in a Transwell system with and without compounds. The co-cultures of hepatocytes were divided into nine groups, i.e., normal, CCl4-treated, three groups of only compound-treated, MSCs-treated and three groups of MSCs + compounds-treated. A significant decrease in the level of LDH was seen in compounds-treated and MSCs + compounds-treated injured hepatocytes ( Figure 1A). However, treatment with MSCs + compounds, predominantly compound (10), showed a significantly higher decrease in cell injury than any other treatment group. Cell viability ( Figure 1B) and GSH level ( Figure 1C) were highly increased in MSCs + compounds (10)-treated hepatocytes. 9.04 ± 0.51 7.3 ± 0.07

In Vitro Enhanced Hepatocytes Survival after Treatment with MSCs + Compounds
First, the hepatocytes were exposed to a CCl 4 dose (5 mM for 6 h) followed by treatment with compounds (10), (14) and (18) for 24 h. After the compounds treatment, MSCs were co-cultured with hepatocytes in a Transwell system with and without compounds. The co-cultures of hepatocytes were divided into nine groups, i.e., normal, CCl 4 -treated, three groups of only compound-treated, MSCs-treated and three groups of MSCs + compoundstreated. A significant decrease in the level of LDH was seen in compounds-treated and MSCs + compounds-treated injured hepatocytes ( Figure 1A). However, treatment with MSCs + compounds, predominantly compound (10), showed a significantly higher de-crease in cell injury than any other treatment group. Cell viability ( Figure 1B) and GSH level ( Figure 1C) were highly increased in MSCs + compounds (10)-treated hepatocytes. Gene expression analysis showed reductions in apoptotic markers such as BAX, caspase-3 and NF-κB in hepatocytes treated with MSCs and compounds alone, as compared to being CCl 4 -treated, but a significant reduction was observed after treatment with MSCs + compounds, particularly compound (10), compared to all other groups. Likewise, an increased level of anti-apoptotic markers BCl 2 was observed after treatment by MSCs or compounds, but a greater increase was observed after the combined treatment of MSCs + compounds (compound (10)), compared to CCl 4 -treated hepatocytes ( Figure 2).  Immunostaining was performed for the detection of caspase-3 in an in vitro co-culture model. The primary antibody used was rabbit polyclonal IgG (1:500, abcam), and the secondary antibody used was goat anti-rabbit IgG (1:1000, abcam). The MSCs + compounds-treated group showed low Casepase-3 protein expression, compared to the MSCs-, only compound-and CCl4-treated groups ( Figure 3A-I). Expression of normal hepatic genes (albumin (A) and CK-8 (B)) and anti-apoptotic (BCl 2 (C)) markers genes was increased and apoptotic (BAX (E), caspase-3 (F) and NF-κB (D)) genes decreased in MSCs + compounds-treated hepatocytes, especially in the case of + MSCs + compound (10)-treated hepatocytes, as shown by bars with symbols *. Mean ± SEM (n = 3). Bars with symbols show significance with each other. * p < 0.02 for MSCs + Comp10 vs. CCl4; # p < 0.05 for MSCs + Comp10 vs. MSCs; ϕ p < 0.05 for MSCs + Comp10 vs. MSCs+Comp18; δ p < 0.05 for MSCs + Comp10 vs. MSCs + Comp14.

Immunostaining for Protein Expression of Caspase-3 in In Vitro Co-Culture Model
Immunostaining was performed for the detection of caspase-3 in an in vitro co-culture model. The primary antibody used was rabbit polyclonal IgG (1:500, abcam), and the secondary antibody used was goat anti-rabbit IgG (1:1000, abcam). The MSCs + compoundstreated group showed low Casepase-3 protein expression, compared to the MSCs-, only compound-and CCl 4 -treated groups ( Figure 3A-I).

Comparative Morphological Study of Liver
All experimental animals were anesthetized; the livers were isolated and the morphology of livers was observed. The comparative morphology can be seen in Figure 4A-F. The morphology of CCl 4 -treated animal liver was more brown in color, with large scars ( Figure 4B). The MSCs-treated animal liver was less brown and scars were decreased ( Figure 4C). The scars gradually decreased in MSCs + compounds-treated groups ( Figure 4D-F), but significant decreases in scars were noted in the MSCs + compound (10)-treated group, and the color of liver was radish, close to the normal liver morphology ( Figure 4F).

Comparative Morphological Study of Liver
All experimental animals were anesthetized; the livers were isolated and the morphology of livers was observed. The comparative morphology can be seen in Figure 4A-F. The morphology of CCl4-treated animal liver was more brown in color, with large scars ( Figure 4B). The MSCs-treated animal liver was less brown and scars were decreased ( Figure 4C). The scars gradually decreased in MSCs + compounds-treated groups ( Figure  4D-F), but significant decreases in scars were noted in the MSCs + compound (10)-treated group, and the color of liver was radish, close to the normal liver morphology ( Figure  4F).  (18), (E) MSCs + compounds (14), (F) MSCs + compounds (10). Liver morphology showed that fibrosis was greatly reduced in MSCs + compound (10)-treated experimental model, with less scars and a radish color, resembling with normal liver.

Homing of Transplanted MSCs in Fibrotic Liver
The MSCs were labeled with PKH67 (green cell-linker dye) and, for the examination of nuclei DAPI, were transplanted into the liver of a fibrotic rat model along with compounds or in the absence of compounds for the detection of the homing of MSCs. The improved homing in fibrotic liver of the models subjected to MSCs + compounds ( Figure  5A (18), (E) MSCs + compounds (14), (F) MSCs + compounds (10). Liver morphology showed that fibrosis was greatly reduced in MSCs + compound (10)-treated experimental model, with less scars and a radish color, resembling with normal liver.

Homing of Transplanted MSCs in Fibrotic Liver
The MSCs were labeled with PKH67 (green cell-linker dye) and, for the examination of nuclei DAPI, were transplanted into the liver of a fibrotic rat model along with compounds or in the absence of compounds for the detection of the homing of MSCs. The improved homing in fibrotic liver of the models subjected to MSCs + compounds ( Figure 5A

MSCs + Compounds Transplantation Effects on Apoptosis
The amount of TUNEL-positive cells in the fibrotic liver considerably decreased in MSCs + compounds (18), (14) and (10)-transplanted groups (Figure 6), when compared with CCl4; with only MSCs-treated groups, the number of tunnel-positive cells significantly decreased in MSCs + compounds (18, 14 and 10)-treated experimental models. Among all groups, the + MSCs + compounds (10)-transplanted group showed a highly decreased number of apoptotic cells ( Figure 6F).

Analysis for Gene Expression after MSCs + Compounds Transplantation
Real-time PCR analysis was performed for all experimental models; the expression of the hepatic, anti-apoptotic and apoptotic genes was studied after 2 weeks of

MSCs + Compounds Transplantation Effects on Apoptosis
The amount of TUNEL-positive cells in the fibrotic liver considerably decreased in MSCs + compounds (18), (14) and (10)-transplanted groups ( Figure 6), when compared with CCl 4 ; with only MSCs-treated groups, the number of tunnel-positive cells significantly decreased in MSCs + compounds (18, 14 and 10)-treated experimental models. Among all groups, the + MSCs + compounds (10)-transplanted group showed a highly decreased number of apoptotic cells ( Figure 6F).

Analysis for Gene Expression after MSCs + Compounds Transplantation
Real-time PCR analysis was performed for all experimental models; the expression of the hepatic, anti-apoptotic and apoptotic genes was studied after 2 weeks of MSCs transplantation. In rats treated with MSCs + compound (10), the expression level of apoptotic (caspase-3, TNF-α, NF-kβ and BAX) genes greatly decreased, and the expression of the liver marker genes (albumin and CK-8) and anti-apoptotic (Bcl-xI and BCl 2 ) genes was up-regulated, compared to CCl 4 -treated and only MSCs-transplanted rat models (Figure 7).

Improved Liver Function after MSCs + Compounds Treatment
Glycogen storage is a key function of hepatocytes. In all experimental groups, PAS-staining was used to assess glycogen storage levels. When compared to MSC alone, MSC + compounds (18), (14) and (10) demonstrated improved glycogen storage recovery. The MSC + compound (10)-treated group showed highly increased recovery of glycogen storage ( Figure 8F).

Biochemical Functions
After two weeks of MSCs transplantation, serum, AST, ALT, ALP and bilirubin levels were compared between the seven experimental rat models to measure liver function. Compared to other animal groups, MSCs + compounds (18)-, (14)-and (10)-implanted rat models had considerably lower levels of enzymes. Among these, MSCs + compound (10)-treated animals showed a highly decrease level of enzymes, compared to the others, as shown in (Figure 9).

Improved Liver Function after MSCs + Compounds Treatment
Glycogen storage is a key function of hepatocytes. In all experimental groups, PASstaining was used to assess glycogen storage levels. When compared to MSC alone, MSC + compounds (18), (14) and (10) demonstrated improved glycogen storage recovery. The MSC + compound (10)-treated group showed highly increased recovery of glycogen storage ( Figure 8F).

Biochemical Functions
After two weeks of MSCs transplantation, serum, AST, ALT, ALP and bilirubin levels were compared between the seven experimental rat models to measure liver function. Compared to other animal groups, MSCs + compounds (18)-, (14)-and (10)-implanted rat models had considerably lower levels of enzymes. Among these, MSCs + compound (10)-treated animals showed a highly decrease level of enzymes, compared to the others, as shown in (Figure 9).

Histopathological Analysis
In rats transplanted with MSCs and MSCs + compounds, liver histology was examined using Masson trichrome staining. Normal liver sections were found to have a structure which lacks fibrosis and inflammation ( Figure 10A), whereas the CCl4-treated model showed fibrosis by the loss of structural integrity and a high amount of deposited

Histopathological Analysis
In rats transplanted with MSCs and MSCs + compounds, liver histology was examined using Masson trichrome staining. Normal liver sections were found to have a structure which lacks fibrosis and inflammation ( Figure 10A), whereas the CCl 4 -treated model showed fibrosis by the loss of structural integrity and a high amount of deposited collagen ( Figure 10B). Compared to the CCl 4 -treated liver ( Figure 10B) and only MSCs-transplanted liver ( Figure 10C), the transplantation of MSCs + compounds dramatically reduced CCl 4induced fibrosis and collagen levels ( Figure 10D-F). For the quantification of data, Image J software was used. The scale was first briefly changed to micrometers, and was then converted to greyscale. Then, the blue-stained collagen was segmented (isolated) using thresholding and the threshold area was measured. collagen ( Figure 10B). Compared to the CCl4-treated liver ( Figure 10B) and only MSCs-transplanted liver ( Figure 10C), the transplantation of MSCs + compounds dramatically reduced CCl4-induced fibrosis and collagen levels ( Figure 10D-F). For the quantification of data, Image J software was used. The scale was first briefly changed to micrometers, and was then converted to greyscale. Then, the blue-stained collagen was segmented (isolated) using thresholding and the threshold area was measured.

Discussion
Liver illness is responsible for over two million fatalities each year globally, with one million of these deaths attributable to cirrhosis complications. Fibrosis of the liver is currently one of the top ten causes of death worldwide [29]. The final phase of liver fibrosis is cirrhosis, during which the architecture of the liver is disrupted and the extracellular matrix is deposited [30]. Whole-organ allograft transplantation is currently an available treatment of last-stage liver diseases. However, due to the paucity of suitable organs, high pricing and surgical challenges, the use of liver transplantation is restricted [6]. The use of MSCs in cell-based therapy, on the other hand, has been shown to be effective in curing liver fibrosis in numerous basic and clinical research works [8]. One of the most important aspects affecting the healing effectiveness of stem cell treatments is the poor survival of cells after transplantation. This is partially due to the transplanted stem cells' incapacity to endure the oxidative stress and inflammatory responses present in the damage site [16].
Increasing the differentiation potential of MSCs is urgently needed in order to convert them into hepatic cells and repair the potential of MSCs to repair the injured tissue or organ by combination therapy.
In our study, MSCs (MSCs) combined with a novel synthetic compound of benzimidazol derivatives treatments were investigated for their capacity to reduce liver fibrosis in experimental models. These compounds were first screened against fibrosis using an in vitro hepatocytes culture, in which these compounds also increased in glutathione (GSH) level, which shows the antioxidant activity of these derivatives. These compounds were first screened in vitro on CCl 4 -induced injured hepatocytes, in which these compounds also increased in glutathione (GSH) level, which shows the antioxidant activity of these derivatives. An in vitro CCl 4 -induced injury culture of rat hepatocytes was prepared for the screening of its compounds and to determine the therapeutic effect of the combination of MSCs and new chemical entities in regard to decreasing liver fibrosis.
Among the eighteen compounds, three compounds were selected for combination therapy with MSCs, on the basis of low IC50 values in LDH assay, trypan blue exclusion assay and increased glutathione level ( Figure S4). Compounds (10), (14) and (18) were active, in comparison to the other compounds of the series. Compound (10) was highly potent, compared to the other compounds. It has previously been reported that glutathione treatment can improve liver functioning [31]. Our results agree with this because the glutathione level increased significantly in our models. Compound (10) is a benzimidazole derivative consisting of a methyl group (-CH3) attached to a benzene ring at position 3, while all the other compounds in this series do not have this group; thus, the high potency of this compound may be due to the presence of this methyl group. In small-molecule medicines, the methyl group is one of the most prevalent carbon fragments. This simplest alkyl group may change both the biological and physical aspects of the molecule and was found in more than 67 percent of the top-selling medications in 2011.
The simple modification of C-H to C-Me boosts a drug candidate's IC50 value by more than 100-fold [32]. Another compound, (14), of the benzimidazol series, consists of three methoxy groups (O-CH3). In pharmacology, the methoxy group, when attached to benzene ring, takes an important and often vital pharmacophoric part. The benzene ring and the methoxy group, during metabolic oxidation, can both produce hydroxy groups. These hydroxy groups increase the solubility of water and the removal of the metabolite, which results in the termination of the activity of the drug due to the pharmacokinetic impact [33]. The methoxy group may increase or decrease the activity of a drug. In this research, due to the presence of methoxy groups, the compound showed a high level of activity. Another compound, (18), also consists of one methoxy group, which showed a high level of activity compared to the other compounds of their series, but a low level of activity compared to compounds (10) and (14). These three compounds were then studied in combination with MSCs using CCl 4 -induced injury both in vitro and in vivo. The injured hepatocytes were co-cultured with these three compounds, with only MSCs and with MSCs + compounds. MSCs + compounds reduced injury, causing a smaller amount LDH release and a higher number of viable cells in this treatment group ( Figure 1A,B).
The real-time PCR analysis of the combined treatment-MSCs + compounds-cocultured model showed a considerable decrease in pro-apoptotic and an increase in antiapoptotic and hepatic gene expressions (Figure 2), compared to the other experimental models. Here, it is reported that a significant decrease in pro-apoptotic and increase in anti-apoptotic and hepatic marker expressions improved liver functioning in the CCl 4treated rat model [21]. The MSCs + + compound (10) showed a significant decrease in the amount of LDH released, increased cell viability and an increased level of GSH/GSSG (Figure 2A-C). The MSCs + compounds treatment of the CCl 4 -induced injury reduced caspase-3 protein expression in a co-culture model when studied by immunohistochemistry (Figure 3). Protein expression is highly reduced in MSCs + + compound (10)-treated models ( Figure 3I). Caspase-3 suppression is connected with reduced hepatic cell damage and death [34].
The valuable influence of the combined MSCs and compound treatment on the survival of hepatocytes in vitro was extended to in vivo to show that these compounds can improve the hepatic milieu and can also increase hepatocytes survival here, resulting in the greater effectiveness of MSCs therapy in fibrotic liver repair. Compounds were injected and then MSCs were transplanted in the liver of a fibrotic model induced by CCI 4 , in vivo. MSCs combined with the compounds increased the homing of MSCs in the injured liver of a CCl 4 -treated rat model. In the present study, it was demonstrated that the transplanted MSCs combined with compounds were considerably better at homing into the injured liver of rat and did not die away after a further 2 weeks of CCI 4 treatment, in comparison to only MSCs-treated animals ( Figure 5).
The results of this study coincide with the results obtained by the increasing antioxidant levels in hUCMSCs, with edaravone being shown to have a big impact on their ability to heal hepatic tissue [16]. Functional analysis demonstrated that MSCs + compoundstreated groups showed considerably decreased serum, ALT, AST, ALP and bilirubin levels in in vivo (Figure 9).
The results obtained in this study show that the restoration of the amount of ALP and bilirubin in serum enable better liver functions [35]. The results of PAS-staining confirmed that the decrease in glycogen level was restored in livers transplanted with MSCs + compounds, compared to CCl 4 -treated and only MSCs-treated animals ( Figure 8).
In the present study, real-time PCR analysis (Figure 7) confirmed that the MSCs + compounds treatment of a CCl 4 -treated injured liver fibrotic model considerably decreased apoptotic (BAX, caspase-3, NF-Kβ and TNF-α) genes expression, and up-regulated antiapoptotic (BCl 2 and Bcl-xL) marker genes. The low expression of apoptotic and high expression of anti-apoptotic markers in the liver may be due to the enhanced therapeutic potential of MSCs combined with compounds. These results coincide with the experiments, which proved that the up-regulation of anti-apoptotic gene Bcl-xL and BCl 2 expression and the down-regulation of BAX, caspase-3, NF-κB and TNF-α are indicative of improved liver functioning [36]. It was also reported that increased antioxidant levels in hUCMSCs with edaravone can have a big impact on their ability to heal hepatic tissue. As shown in the literature, hepatic functions were improved and the host liver was regenerated [16].

Isolation and Culturing of Rats Hepatocytes
Hepatocytes were isolated from male Wister rats using a modified in situ collagenase perfusion method [26]. The cell viability of the hepatocytes cell, which was isolated from rats, was measured through a trypan blue exclusion test. Then, the hepatocytes cells were isolated; cultured in collagen coated plates in an RPMI medium; augmented with 100 Units/mL of penicillin, 100 µg/mL of streptomycin, 50 ng/mL of EGF and 10% FBS; and then incubated at 37 • C in a 5% CO 2 atmosphere. The morphology of the isolated cells was examined after 2 h and 24 h of culturing, under a phase-contrast light microscope, for the confirmation of their hepatic origin. RNA was extracted from the cultured cells using an E.Z.N.A. ® RNA Isolation Kit procedure, and the cDNA was synthesized using a cDNA Synthesis Kit. One µI of cDNA generated from the cultured hepatocytes was used for the RT-PCR analysis of specific markers (CK-8, CK-18 and albumin) of hepatocytes. Primer size and sequences are given in Table 1. As an internal control, β-Actin was used. Immuno-cyto chemistry was used for cultured cells to determine specific markers (albumin, CK-18 and CK-8) and their expression of hepatocytes at a protein level. A PAS-staining test was performed for glycogen storage to examine whether the cultured cells were functionally active hepatocytes.

In Vitro CCl 4 -Induced Injury of Hepatocytes
In vitro CCl 4 -induced injury to hepatocytes was given by treating the cultured hepatocytes with a 5 mM concentration of CCl 4 in DMSO, as described previously [27]. Non-treated cultured hepatocytes were considered normal. After 6 h of injury with CCl 4 , the media from the cultured cells were collected and stored for the LDH assay.

Lactate Dehydrogenase (LDH) and Cell Viability Assay
The hepatocytes medium was centrifuged at 250× g for 4 min, and the LDH activity of the supernatant was evaluated at 492 nm, according to the kit protocol (Roche). The cell viability assay for CCl 4 -treated injured cells, compound-treated, MSCs-treated and + MSCs + compounds-treated hepatocytes cells was performed using a trypan blue-exclusive methodology. The number of viable cells was counted by dividing the number of trypan blue negative cells by the total number of cells observed and multiplying this by 100.

GSH/GSSH Assay
The glutathione assay was performed for the CCl 4 -treated injured cells, compoundtreated, MSCs-treated and MSCs + compounds-treated hepatocytes cells, according to the kit's procedure (GSH/GSSG-Glo™ Assay kit, promega); briefly, the medium containing the test compounds was removed from cells. Both the lysis reagent were prepared and added into each well; then, the plates were shacked, and 50 µL of luciferin generation reagent was added to each well, shook for 30 min, and then, 100 µL of luciferin detection reagent was added to each well. The plates were shook again; after waiting for 15 min, the luminescence was measured using a Tecan Microplate Reader (Tecan Group Ltd., Männedorf, Zürich, Switzerland).

Immunostaining
Immunostaining was conducted to find the expression of caspase-3 protein in normal hepatocytes, CCl 4 -treated and compounds-treated, MSCs-treated and MSCs + compoundstreated hepatocyte. First, the cells were washed 3 times with PBS, each for 5 min, fixed in 4% formaldehyde for 30 min at room temperature and permeabilized with 0.1% triton for 10 min; then, nonspecific binding was inhibited by blocking with 2% BSA in PBS for 45 min. The cells were then incubated with primary antibodies for 4 h at room temperature. The primary antibody used for caspase-3 was rabbit monoclonal (1:1000, abcam) and the secondary antibody was goat anti-rabbit (1:500, abcam, Cambridge, UK). The samples were incubated for 45 min at room temperature (RT), and then were washed with PBS and incubated with DAPI for 2-5 min at room temperature. Then, the samples were again washed with PBS, and subsequently mounted with vecta sheets. The samples were then observed using a Fluoview FV 3000 microscope (Olympus, Tokyo, Japan) and pictures were taken.

MSCs Isolation and Culturing
MSCs were isolated from the femur bone and tibia bone of Wister rats that were 250 g-300 g in weight according to the protocol previously described [36]. The cells were cultured in a 25 cm 2 culture flask in IMDM medium, which was supplemented with 20% fatal bovine serum (FBS), 100 µg/mL of streptomycin and 100 U/mL of penicillin, and incubated at a temperature of 37 • C in an atmosphere containing 5% CO 2 . On the third day, the culture medium was changed to plated cells. For the confirmation of the existence of the MSCs and, in order to remove hematopoietic stem cells, FACS analysis was performed. CD90, CD44, CD105 and CD34 antibodies were used.

In Vitro Injured Hepatocytes Co-Culture Model
In vitro injury was given for 6 h by treating cultured hepatocytes with a 5 mM concentration of CCl 4 in DMSO, as described previously [27]. The co-cultured model was established by culturing injured hepatocytes treated with MSCs and compounds (18), (14) and (10) for 24 h in a Transwell culture system with DMEM (sigma) medium containing 10% FBS, 100 µg/mL of streptomycin and 100 U/mL of penicillin. The co-culture was undertaken using a porous Transwell membrane, with a pore size of 0.4 mm (BD Biosciences). Hepatocytes were first seeded at a density of 1.5 × 10 5 /cm 2 on the collagen-coated 6-well plate. Once attached, the MSCs were seeded onto the Transwell membrane inserts at a density of 1.5 × 10 4 . The hepatocytes were divided into six groups: normal, CCl 4 -treated injured, CCl 4 -treated injured co-cultured with MSCs alone, CCl 4 -treated injured co-cultured combined with MSCs + compound (18), CCl 4 -treated injured co-cultured combined with MSCs + compound (Luciferin Generation Reagent) and CCl 4 -treated injured co-cultured combined with MSCs + compound (10). After 24 h of co-culturing, the treated hepatocytes cells were then collected for the extraction of their RNA, LDH cytotoxicity assay, trypan blue assay, glutathione assay and immunostaining.

Gene Expression Analysis
From the CCl 4 -treated injured cells and other treated cells, and also from liver tissues samples, RNA was extracted using an E.Z.N.A. ® Total RNA kit, according to the kit protocol. The quantification of RNA was conducted using a Spectrophotomter Nano drop; ND-1000 and cDNA were synthesized by a BIO-RAD I Script tm cDNA synthesis kit using RT-PCR.
Real-time PCR analysis was carried out with SYBR Green PCR Super Mix (Bio-Rad, Hercules, CA, USA) and 2 µI of cDNA. The procedure of PCR consisted of an initial period of denaturation at a temperature of 94 • C for 4 min, 35 cycles of denaturation for 45 s and then annealing at 56 • C-58 • C for 45 s and an extension at 72 • C for 45 s, followed by a final step of extension at 72 • C for 10 min. The expression levels of albumin, cyto-keratin 8, Bcl 2 , BAX, Bcl-xL, TNF-α, NF-κβ and caspase-3 were measured. The comparative CT method (∆∆Ct value) was used to measure the relative expression of target genes. As a reference, gene β-actin was used. All primer sequences are given in Table 2.

CCl 4-Induced LiverFibrotic Model
Male Wister rats were used for the liver fibrotic model. Rats were kept with free access to water and food in sterilized cages. For the preparation of the model, 1 µL/g of body weight of CCl 4 (1:1 in olive oil) was injected intraperitoneally for four weeks, twice per week, as described previously [27].

In Vivo Compounds Treatment
After the completion of 4 weeks of CCl 4 treatment, compounds (18), (14) and (10) were injected at a dose of 50 µg/kg intraperitoneally. The rats were divided into seven groups: normal, CCl 4 , MSCs and two groups of MSCs + compounds-treated rats; each experimental group consisted of six animals.

MSCs Transplantation
Cultured MSCs were trypsinized and then labelled with PKH-67 (green) cell linker fluorescent dye (Sigma-Aldrich, St. Louis, MO, USA), as described previously [37]. MSCs were transplanted to MSCs + compounds groups for four hours after injecting compounds. All groups were euthanized, and the abdomen was cut to expose the liver. Approximately 10 7 cells were transplanted in 1 mL of PBS into a liver lateral and median lobes using a 30 G syringe.

Periodic Acid Schiff (PAS) Assay
After 15 days of the MSCs and compounds treatment, PAS-staining was performed for all experimental groups for the measurement of glycogen storage levels in liver sections. First, the 5 µm thick microtome sections of the liver were de-paraffinized. Tissue sections were then incubated for 5 min in periodic acid at RT. Then, tissue sections were washed with distilled H 2 O and stained with Schiffs reagent for 15 min and then hematoxylin-stained for 90 s. Tissue sections were then washed with tap water; the sections were then mounted and observed under microscope.

Masson Trichrome-Staining
The formalin-fixed liver tissue was dehydrated and then embedded in paraffin. The tissue was cut into 5 µm thick sections by a microtome Leica RM 2155 (Leica Biosystems, Wetzlar, Germany) and slides were prepared. Then, they were stained for the collagen using a Masson trichome staining kit (Abcam), according to the kit protocol. Collagens were observed under an Olympus BX-61 microscope in the fibrotic liver and pictures were captured with a Digital Camera camera (Olympus digital camera DP70, Olympus Optical Co., Ltd., Tokyo, Japan). Using Image J software, the quantification of collagen percentage was conducted regarding the stained liver sections [27].

Biochemical Analysis
Blood samples were collected and centrifuged for each experimental group at 8000 rpm for 15 min in order to isolate the serum. Bilirubin, alanine transaminase (ALT), alkaline phosphatase (ALP) and aspartate transaminase (AST) testing is a common group of tests used to check liver health. High levels of ALT, ALP, AST and bilirubin release in blood may be a sign of a liver injury or disease. ALT, ALP, bilirubin and AST levels were measured in serum, according to kit (Centronics, Wartenberg, Germany) protocol.

TUNELAssay for Hepatic Apoptosis
The TUNEL assay, an appropriate tissue processing technique, is a relatively fast, reproducible and quantitative method for detecting apoptosis in tissue. The TUNEL assay was performed using a TUNEL Apoptosis Detection Kit (Merck-millipore, Temecula, CA, USA) for the detection of apoptosis in each group. The 5 µm thick liver sections in paraffin were used in the TUNEL assay. Three sections were selected for each rat. An Olympus BX-61 microscope was used for the examination of the tissues of the experimental groups, and images were taken with a Digital Camera DP-70 (Olympus, Japan). The number of apoptotic hepatocytes was calculated in each section.

Statistical Analysis
All data are presented as mean ± SD. The analysis of fibrosis percentage between groups was performed using one-way ANOVA. The graphs were made with the help of Graph Pad prism 7. p value < 0.05 was considered significant.

Conclusions
In the present study, it is demonstrated that, based on the IC50 values found, compounds (10), (14) and (18) were active against liver fibrosis. These three compounds also increased the glutathione (GSH/GSSG) level in an in vitro experimental model, and can be used as an antioxidant. Furthermore, these compounds increased the potential of MSCs for liver fibrosis reduction. In conclusion, compound (10) was the most potent among all the compounds, can be used as a lead compound for the synthesis of drugs against liver fibrosis, and the potential of the use of MSCs against liver fibrosis is enhanced using these compounds ((10), (14) and (18)) combined with MSCs. MSCs combined with compound (10) significantly improved the liver functions. Here, we report a different therapeutic approach, combining synthetic compounds with MSCs for the treatment of liver fibrosis.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ph16020306/s1, Figure S1. Morphology of rat hepatocytes cells in in vitro culture isolated from male Wister rat: (A) After 2 h of culturing, the cultured cells were spherical in shape and were bi-nucleated in phase-contrast microscope. (B) After 24 h of culturing, hepatocytes were polygonal in shape and were bi-nucleated, showing the morphology of normal hepatocyte (20×; scale bar: 100µm).